8  Microglial subpopulations

Identification of Microglial Subpopulations

Published

August 8, 2025

suppressPackageStartupMessages({
library(tidyverse)
library(Seurat)
library(patchwork)
library(kableExtra)
    })

load("data/20250620-seurat_integrated_UMAP_nFeat-ScTypeannotated.Rdata")
alldata$ribo_percent <- PercentageFeatureSet(alldata,assay = "SCT", pattern = "^Rp[ls]")

8.1 UMAP

Comparison of UMAPs previously annotated using ScType and clustered with Seurat.

Idents(alldata) <- alldata@meta.data$sctype_classification
d1 <- DimPlot(alldata, label = TRUE, repel = T) + 
    NoLegend() + ggtitle("ScType annotated")

Idents(alldata) <- alldata@meta.data$seurat_clusters
d2 <- DimPlot(alldata, label = TRUE, repel = T) + 
    NoLegend() + ggtitle("Seurat clusters")

d1|d2

8.2 Subclustering of Microglial population based on ScType

microglia <- subset(alldata, subset = sctype_classification =="Microglial cells", idents = alldata$seurat_clusters)
microglia$jm = if_else (microglia$seurat_clusters%in%c("14","15","17","22","23"),microglia$seurat_clusters,"bigblob")

Idents(microglia) <- microglia$jm
d3 <- DimPlot(microglia, label = TRUE, repel = T) + 
    NoLegend() + ggtitle("Only microglial cells")

d4 <- FeaturePlot(microglia, features = c("ribo_percent"))

d3|d4

Ribosomal Content Varies Across Microglial Subclusters. However, when examining the top marker genes obtained from FindAllMarkers, there are no ribosomal genes among the most discriminative features. This suggests that while ribosomal content varies, it does not directly drive cluster identity at the transcriptional marker level.

8.3 Cluster-specific markers in microglia

From the previous Find Markers page, using all cells and all clusters

8.3.1 Cluster 22

markers <- read_csv("data/20250728-all_markers.csv")
top <- markers |>
    filter(pct.1>0.3) |> 
    filter(cluster == 22) |> 
    top_n(20, avg_log2FC) |> 
    pull(gene)
## 20 most enriched 
# cat(top, sep = ",")
Gene Broad Class Finer Subtype Notes
Mrc1 Microglia / Macrophage Perivascular macrophages CD206; enriched in border-associated macrophages (BAMs) and perivascular/meningeal macrophages.
F13a1 Microglia / Macrophage Perivascular macrophages Co-expressed with Mrc1; clotting factor XIII subunit A.
Ms4a6c Microglia Homeostatic microglia Member of MS4A family; enriched in microglia under steady-state conditions.
Pf4 Microglia / Macrophage Border-associated macrophages Also known as platelet factor 4; found in BAM-like cells.
Pid1 Microglia Activated microglia Linked to metabolic regulation and activation states.
Dab2 Microglia / Macrophage BAM-like macrophages Endocytic adaptor; enriched in perivascular/meningeal macrophages.
Ctsc Myeloid (Microglia / Macrophage) Activated microglia/macrophages Cathepsin C; lysosomal protease.
Stab1 Microglia / Macrophage Perivascular macrophages Scavenger receptor; strong BAM marker.
Ccl7 Microglia / Macrophage Activated/chemotactic state Chemokine induced by inflammation.
Pim1 Microglia Activated microglia Serine/threonine kinase; inflammatory regulation.
Rnf150 Microglia Homeostatic microglia E3 ubiquitin ligase; enriched in resting microglia.
Ifitm2 Myeloid / Endothelial Interferon-stimulated microglia Broad interferon response gene; can be endothelial too.
Iqgap1 Broad Ubiquitous but higher in activated microglia & astrocytes Scaffold protein; upregulated in injury.
Maf Microglia / Macrophage BAM-like macrophages Transcription factor important for BAM identity.
Ccl8 Microglia / Macrophage Activated state Chemokine; inflammatory signature.
Klf6 Microglia / Astrocyte Activated microglia/astrocytes Injury-induced transcription factor.
Cebpb Microglia / Astrocyte Inflammatory state Regulates reactive glia.
Man1a Broad Housekeeping Golgi alpha-mannosidase; not cell-type specific.
Cytip Microglia / Macrophage BAM-like macrophages Cytokine-induced protein in myeloid cells.
Srgn Microglia / Macrophage Activated myeloid cells Proteoglycan; high in inflammatory myeloid cells.

Almost all these genes map to myeloid lineage in the brain, especially border-associated macrophages (BAMs) — which include perivascular, meningeal, and choroid plexus macrophages — and to a lesser extent activated microglia. The signature is strongly enriched for perivascular macrophage markers (Mrc1, F13a1, Stab1, Pf4, Maf, Dab2) combined with inflammatory chemokines (Ccl7, Ccl8, Cebpb).

8.3.2 Cluster 23

top <- markers |>
    filter(pct.1>0.3) |> 
    filter(cluster == 23) |> 
    top_n(20, avg_log2FC) |> 
    pull(gene)
## 20 most enriched 
# cat(top, sep = ",")
Gene Broad Class Finer Subtype Notes
Ndufa4l2 Endothelial Capillary / hypoxia-responsive EC Mitochondrial complex I subunit; enriched in brain endothelial cells under low oxygen.
Egflam Endothelial / Pericyte Vascular leptomeningeal cells Extracellular matrix glycoprotein; strong in vascular interfaces.
Gucy1b1 Endothelial Arterial/venous EC Soluble guanylate cyclase β1 subunit.
Il34 Neuron (source) / Microglia (target receptor CSF1R) Secreted trophic factor Neurons produce IL-34 to support microglia survival; expression in cortical excitatory neurons.
Arhgap42 Endothelial / Smooth muscle Vascular SMC/pericytes Rho GTPase-activating protein.
Galnt17 Endothelial / Pericyte Vascular interface Glycosyltransferase; enriched in mural cells.
Lin7a Neuron Excitatory neuron (layer-specific) Synaptic scaffolding protein.
Ctdspl Neuron Broad neuronal expression Transcriptional repressor; high in excitatory neurons.
P2ry14 Endothelial / Pericyte Venous EC / pericytes UDP-glucose receptor; enriched in vascular cells.
Sntb1 Neuron / Glia Synaptic protein Found in neurons and astrocytic endfeet; part of dystrophin complex.
Art3 Endothelial / Pericyte Vascular smooth muscle-like ADP-ribosyltransferase; mural cell enrichment.
Airn Endothelial Capillary EC (non-coding RNA) Imprinted lncRNA; vascular expression.
Myl9 Smooth muscle / Pericyte Contractile pericytes Myosin light chain 9.
Ecm2 Endothelial / Pericyte Extracellular matrix of vasculature ECM component; vascular specificity.
Nbl1 Endothelial Vascular niche BMP antagonist; enriched in EC.
Septin11 Broad Cytoskeletal; ubiquitous Not cell-type specific; structural role.
Kcnj8 Pericyte / SMC Contractile mural cells Potassium inward rectifier channel Kir6.1.
Cspg4 Pericyte / OPC NG2+ cells Canonical marker of pericytes and oligodendrocyte precursor cells.
Ddit4l Endothelial / Astrocyte Hypoxia-stress responsive Regulates mTOR; induced in vascular and glial cells under stress.
Morrbid Myeloid (perivascular macrophages) Apoptosis regulator lncRNA in tissue-resident myeloid cells; in brain, enriched in BAMs.

This set is dominated by vascular-associated cells — mostly endothelial cells and mural cells (pericytes, vascular smooth muscle cells) — with a few neuronal genes (Il34, Lin7a, Ctdspl), one perivascular macrophage gene (Morrbid), and a dual-identity pericyte/OPC marker (Cspg4). The strong enrichment for Ndufa4l2, P2ry14, Kcnj8, Myl9 points to capillary/perivascular structures, often at the brain–blood barrier.

8.3.3 Cluster 15

top <- markers |>
    filter(pct.1>0.3) |> 
    filter(cluster == 15) |> 
    top_n(20, avg_log2FC) |> 
    pull(gene)
## 20 most enriched 
# cat(top, sep = ",")
Gene Broad Class Finer Subtype Notes
Itk Immune (T cell) T lymphocytes IL-2–inducible T-cell kinase; canonical T-cell signaling.
Cd3g Immune (T cell) T lymphocytes CD3 complex γ chain; TCR signaling.
Cd3e Immune (T cell) T lymphocytes CD3 complex ε chain; core T-cell receptor component.
Grap2 Immune (T cell/NK) T cells, NK cells Adaptor in TCR/NK signaling.
Cxcr6 Immune (T cell) Tissue-resident memory T cells Chemokine receptor guiding brain TRM infiltration.
Themis Immune (T cell) T lymphocytes Thymocyte development protein.
Gimap4 Immune (T cell) T lymphocytes Small GTPase enriched in T cells.
Lck Immune (T cell/NK) TCR/NK signaling Src-family kinase in TCR signaling.
Trac Immune (T cell) αβ T lymphocytes TCR α constant region.
Cd226 Immune (T cell/NK) Cytotoxic T cells, NK cells Co-stimulatory receptor.
Stat4 Immune (T cell/NK) Th1 CD4⁺ T cells, NK cells Transcription factor in IL-12 signaling.
Atp8b4 Microglia / Myeloid Homeostatic microglia Phospholipid transporter; strong microglial marker in brain.
Ikzf3 Immune (B cell/T cell) Memory B cells, T cells Aiolos transcription factor.
Ctsw Immune (T cell/NK) Cytotoxic lymphocytes Cathepsin W; granule protein in CD8⁺ T cells and NK cells.
Runx3 Immune (T cell) CD8⁺ cytotoxic T cells Lineage-defining transcription factor.
Adam19 Broad Various Metalloproteinase; expressed in multiple lineages, higher in vascular cells.
Pde7a Immune (T cell) T lymphocytes cAMP phosphodiesterase enriched in T cells.
Gramd3 Broad Limited immune association Poorly characterized; low-level in immune and vascular cells.
Bcl2 Broad Anti-apoptotic Expressed in many cell types; survival factor in lymphocytes.
Ptprc (CD45) Immune (pan-leukocyte) All immune cells Canonical pan-leukocyte marker.

This list is overwhelmingly T-cell–specific, with strong enrichment for CD3-complex genes (Cd3e, Cd3g, Trac), TCR signaling molecules (Itk, Lck, Themis, Grap2), and effector/memory T cell markers (Cxcr6, Runx3, Cd226, Stat4). These signatures correspond to brain-infiltrating T lymphocytes, especially CD8⁺ cytotoxic and tissue-resident memory (TRM) subsets, with a possible small contribution from NK cells.

One gene (Atp8b4) is clearly a microglia marker, suggesting either minor contamination or genuine presence of myeloid cells in this population. Ptprc confirms leukocyte origin.

8.3.4 Cluster 17

top <- markers |>
    filter(pct.1>0.3) |> 
    filter(cluster == 17) |> 
    top_n(20, avg_log2FC) |> 
    pull(gene)
## 20 most enriched 
# cat(top, sep = ",")
Gene Broad Class Finer Subtype Notes
Syngr1 Neuron Broad neuronal (synaptic vesicle protein) Pan-synaptic marker; in both excitatory and inhibitory neurons.
Tyrobp Microglia / Macrophage Activated microglia, DAM (disease-associated microglia) Adaptor protein in TREM2 signaling.
Fau Broad Housekeeping Ribosomal protein; not cell-type specific.
Rplp1 Broad Housekeeping Ribosomal large subunit protein; ubiquitous.
Tmsb4x Broad (with immune bias) Cytoskeleton/actin regulator Expressed in many cells; high in microglia and astrocytes.
Fcer1g Microglia / Macrophage DAM/activated microglia Immunoreceptor adaptor in TREM2–TYROBP axis.
Trem2 Microglia DAM signature Alzheimer’s and injury-associated microglia.
Cst3 Microglia / Astrocyte Broad glial Cystatin C; high in microglia, astrocytes, and choroid plexus epithelium.
Cd52 Immune (lymphoid/myeloid) Lymphocytes, some microglia GPI-anchored protein; leukocyte marker with low microglial expression.
C1qc Microglia Homeostatic microglia Complement subcomponent; part of C1q complex.
Ctsd Microglia / Broad glia Lysosomal protease High in microglia and astrocytes.
C1qa Microglia Homeostatic microglia Complement component; C1q complex.
Ctsz Microglia Activated microglia Lysosomal protease upregulated in activation.
C1qb Microglia Homeostatic microglia Complement C1q complex.
Ctsb Microglia / Broad glia Lysosomal protease Ubiquitous in glia; higher in microglia.
Hexb Microglia Canonical microglia marker Beta-hexosaminidase subunit; stable homeostatic marker.
Cox6a2 Neuron Oxidative phosphorylation, neuronal subtype Mitochondrial protein; often higher in fast-spiking neurons.
Grn Microglia / Astrocyte Activated microglia Progranulin; injury/inflammation marker.
Fxyd5 Broad (Glia bias) Astrocytes, microglia Cell adhesion regulator.
Mpeg1 Microglia / Macrophage BAMs and activated microglia Perforin-like protein; high in phagocytic myeloid cells.

This list is dominated by microglia, with a strong homeostatic core signature (C1qa, C1qb, C1qc, Hexb) combined with activation/DAM markers (Trem2, Tyrobp, Fcer1g, Grn, Mpeg1). This suggests you are seeing a mixed microglial population — mostly homeostatic, with a substantial activated/DAM subset.

A few genes are neuronal (Syngr1, Cox6a2) or ubiquitous ribosomal (Fau, Rplp1), which could represent low-level neuronal contamination or ambient RNA.

8.3.5 Cluster 14

top <- markers |>
    filter(pct.1>0.3) |> 
    filter(cluster == 14) |> 
    top_n(20, avg_log2FC) |> 
    pull(gene)
## 20 most enriched 
# cat(top, sep = ",")
Gene Broad Class Finer Subtype Notes
Ifi211 Myeloid (microglia/macrophage) Interferon-stimulated microglia Part of IFN-inducible family.
Ifit3 Myeloid (microglia/macrophage) Type I IFN response Canonical antiviral gene.
Mx1 Myeloid (microglia/macrophage) IFN-stimulated antiviral state Dynamin-like GTPase; viral defense.
Ifi209 Myeloid IFN-stimulated microglia AIM2-like receptor family.
Ifit2 Myeloid IFN-induced microglia Antiviral IFN response protein.
A330040F15Rik Myeloid IFN-response Predicted gene; co-expressed with ISGs.
Oasl2 Myeloid IFN-stimulated antiviral 2’-5’-oligoadenylate synthetase-like.
Rsad2 (Viperin) Myeloid Antiviral IFN response Strong ISG in microglia/macrophages.
Ifi204 Myeloid IFN/DNA-sensing microglia p204 protein, innate immunity.
Gm4951 Myeloid IFN-induced Predicted; co-expression with ISGs.
Ccl12 Myeloid Activated inflammatory macrophages Monocyte chemoattractant.
Parp14 Myeloid IFN-stimulated ADP-ribosyltransferase, antiviral role.
Herc6 Myeloid IFN-induced antiviral E3 ligase in ISGylation pathway.
Ifih1 Myeloid Viral RNA sensing microglia MDA5 helicase; innate immune sensor.
Bst2 Myeloid IFN-induced microglia/endothelial Tetherin; antiviral restriction factor.
Pik3ap1 Myeloid / B cell B cell receptor adaptor; some myeloid expression Possible infiltrating lymphoid origin.
Cxcl10 Myeloid IFN-induced chemokine T cell chemoattractant (IP-10).
Lyn Myeloid / B cell Src-family kinase in immune signaling Present in microglia and B cells.
Slc15a3 Myeloid IFN-induced microglia/macrophages Lysosomal peptide transporter; innate immunity.
Hpse Myeloid / Endothelial Activated immune cells Heparanase; ECM remodeling during inflammation.

This is a Type I interferon–driven microglial/macrophage activation signature with strong antiviral and inflammatory chemokine components. Key canonical ISGs (Ifit3, Mx1, Rsad2, Oasl2, Cxcl10) are all upregulated in microglia during viral infection models, neuroinflammation, or interferonopathies.

A few genes (Pik3ap1, Lyn) have higher expression in B cells, so there might be a minor infiltrating B-lymphocyte component, but the dominant source here is clearly microglia in an interferon-activated state.

8.4 Treatments

All those cluster appear in both treatment groups:

DimPlot(microglia, label = TRUE, repel = T, split.by = "type") + 
    NoLegend()